Cell-based therapies are emerging as a potentially powerful treatment for osteogenesis imperfecta, a genetic skeletal disorder characterized by bone fragility and deformities caused by defects in type I collagen. 0steogenic progenitor cells of possible therapeutic utility exist in skeletal muscle tissue, although their identity is unknown. In the present proposal, in vivo bone induction assays in mice will be used in conjunction with Cre/lox lineage tracing to definitively establish the osteogenic potential of five candidate cell types resident in skeletal muscle tissue: quiescent and activated muscle satellite cells, vascular smooth muscle (VSM) cells, pericytes and endothelial cells. Each cell type will be specifically and permanently labeled with lacZ by intercrossing two mouse strains: transgenic mice that express the gene for Cre recombinase under the control of a cell type-specific promoter/enhancer (MyoD-cre for satellite cells, smooth muscle myosin heavy chain-cre for VSM and pericytes, and Tie2-cre for endothelium) and Rosa26 Cre reporter mice, in which LacZ expression is Cre-dependent. The osteogenic potential of these cell types will be determined by following their fate after intramuscular injection of purified bone morphogenetic protein 2, which induces a robust osteogenic response that recapitulates normal endochondral bone formation. Cellular contributions to cartilage and bone will be determined by 0-galactosidase histochernistry and immunohistochemistry using cell-specific markers. In addition, the genetic control of satellite cell commitment and osteoinduction will be explored by assessing the osteogenic potential of satellite cells in mice carrying a null mutation in the muscle regulatory gene, MyoD. Finally, the osteogenic potential of muscle side population (SP) stem cells-recently identified by FACS analysis-will be evaluated in cell culture, and their relationship to satellite cells, VSM, pericytes and endothelium will be determined by combining lineage marking with FACS analysis of muscle-derived cells. Establishing the identity of osteogenic progenitor cells of skeletal muscle will provide the foundation for investigating mechanisms regulating commitment to the osteogenic lineage. Evaluating these osteoprogenitor cells in future cell transplantation models will provide an essential preclinical test of their utility for the treatment of osteogenesis imperfecta and other diseases of the musculoskeletal system.